Original article Water relations of spruce seedlings sprayed with a surfactant M Borghetti Istituto Miglioramento Genetico delle Piante Forestali, Consiglio Nazionale delle Ricerche, via S Bonaventura, 13-50145 Florence, Italy (Received 14 August 1990; accepted17 January 1991) Summary — This work assesses whether the water relations of Norway spruce (Picea abies Karst) seedlings are affected by repeated treatments with ABS (sodium dodecylbenzensulphonate). Begin- ning in August 1987 4-yr-old spruce seedlings were sprayed weekly with a 50 g m -3 ABS solution. Two experiments were performed in 1988 and 1989, during which water potential, transpiration rates and stomatal conductance were measured on treated and control plants, which were either well- watered or exposed to periods of drought of different duration. Net photosynthesis and specific leaf area of needles were also measured. ABS did not alter transpiration or stomatal conductance asso- ciated with xylem water potential and had no effect on net photosynthesis and specific leaf area. Picea abies / Pinaceae / Norway spruce / surfactant / pollution / transpiration / stomatal con- ductance / water potential Résumé — Relations hydriques des plantules d’épicea commun pulvérisées avec un agent tensio-actif. Ce travail a été conduit afin d’évaluer si des traitements avec des solutions d’ABS (do- decylbenzensulphonate de sodium) influencent les relations hydriques des plantules d’épicea (Picea abies Karst). Pour cela, des plantules âgées de 4 ans ont été pulvérisées, toutes les semaines à partir d’août 1987, avec une solution d’ABS à la concentration de 50 g m -3 . Deux expériences ont été réalisées, en 1988 et 1989, pendant lesquelles on a mesuré le potentiel hydrique, la conduc- tance stomatique et la vitesse de transpiration des plantules, les unes irriguées, les autres soumises à des périodes de sécheresse de durées différentes. On a mesuré aussi la photosynthèse nette et la surface foliaire spécifique des aiguilles. L’ABS n’a pas alteré la transpiration et la conductance sto- matique associées avec le potentiel hydrique et n’a pas eu effet sur la photosynthèse nette et la sur- face foliaire spécifique. Picea abies / Pinaceae / épicea / tensio-actif / pollution / transpiration / conductance stomati- que / potentiel hydrique INTRODUCTION In recent years it has been proposed that air pollutants may directly affect leaf me- tabolism and vitality, playing a role in the so-called "forest decline" phenomenon (Schütt and Cowling, 1985; Karhu and Huttunen, 1986; Sauter and Voss, 1986). Indeed, alterations of leaf surfaces by at- mospheric pollutants have been demon- strated in conifer and broadleaf trees. These findings concern both field observa- tions and experiments carried out under controlled conditions, the most frequently described effect being the degradation of the epicuticular waxes in the stomatal an- techamber (Percy and Riding, 1978; Cape and Fowler, 1981; Huttunen and Laine, 1983; Crossley and Fowler, 1986; Rinallo et al, 1986; Sauter and Voss, 1986; Sauter et al, 1987; Barnes et al, 1988; Raddi and Rinallo, 1989; Rinallo and Raddi, 1989a). The effect of pollutants on wax morpholo- gy of conifer needles has been reviewed by Riederer (1989). The physiological importance of surface waxes on leaves is well known (Hall and Jones, 1961). The observed morphological alterations may support the idea that air pollutants have the capacity to alter the transport properties of the cuticle and im- pair the gaseous diffusional processes. For instance, in conifers the wax-filled stomatal antechamber contributes two- thirds of the resistance to the water vapour diffusion (Jeffree et al, 1971) and it is rea- sonable to suppose that this resistance may increase after the degradation of the so-called structural waxes into a more amorphous and less porous material. Recently, alterations of the epicuticular waxes following treatments at low concen- tration with a surfactant (sodiumdodecyl- benzensulphonate, ABS), which is an im- portant component of synthetic detergents, have been described in broadleaf seed- lings (Rinallo and Raddi, 1989b). ABS was also demonstrated to have a role as water and air pollutant and is supposed to greatly contribute to the decline of forest coastal vegetation in Tuscany, Italy (Gellini et al, 1983, 1985). For these reasons, research has been carried out to determine whether or not there is a change in the water relations of spruce (Picea abies Karst) seedlings sub- jected to repeated treatments with ABS at low concentration. In the area where the study has been performed ABS concentra- tions similar to those used in the present work can be detected (Rinallo and Raddi, 1989b). MATERIALS AND METHODS Plant material and treatments Four-yr-old potted spruce seedlings of alpine provenance (Val di Fiemme, Trentino, Italy) were maintained in the glasshouse. From Au- gust 1987, 100 randomly sampled seedlings were sprayed weekly until the leaves were satu- rated to drip point with a 50 g m -3 ABS (sodium dodecylbenzensulphonate with a non linear alkyl group) aqueous solution. Another lot of 100 ran- domly sampled seedlings was used as control plants. Experiment 1 The plants used in this experiment underwent a minimum of 35 and a maximum of 50 treatments with ABS solution. The needles produced in spring 1988 (current needles) had undergone between 4-12 treatments at the time of measur- ment. The main objective in this experiment was to study whole plant transpiration and stomatal conductance under a wide range of plant water status, which was obtained by imposing periods of drought of different duration. In spring 1988 the ABS-treated and the control plants were di- vided into 2 groups (A and B) of 50 plants each, which underwent different irrigation schedules (fig 1). Irrigation was carried out by supplying the same amount of water to each pot every time. Measurements of xylem water potential, whole plant transpiration and stomatal conduc- tance were carried out at different dates (fig 1). Measurements were performed simultaneously on each plant, approximately every 2 h from 7:00 h am to 6:00 h pm. Three plants per group were used for measuring water potential and 3 more plants for measuring transpiration and stomatal conductance. Different series of plants, randomly sampled from group A and B, were measured at each date. On August 3 1988, net photosynthesis was also measured at 3 different times of day. The xylem water potential was measured with a pressure chamber on 1-yr-old twigs, sam- pling 1 twig per plant. The whole plant transpira- tion was measured by weighing the plants at regular intervals to the nearest 0.1 g using a top-loading balance, after sealing the pots in plastic bags to prevent water loss from the soil. The stomatal conductance and net photosynthe- sis were measured respectively with a null- balance steady state porometer (Li-1600, Li-Cor Inc, Lincoln, Nebraska, USA) and with an IRGA portable open system (LCA2, ADC Ltd, Hodde- son, Herts, UK), on 3 current twigs per plant. The leaf area of entire seedlings and meas- ured twigs was determined at the end of each measurement day, using an optical area meter (Li-3000, Li-Cor Inc, Lincoln, Nebraska, USA). The specific leaf area of both ABS-treated and control plants was determined as the ratio between the projected leaf area and the needle dry weight; dry weight was measured to the nearest 0.1 mg after 48 h in an oven at 60 °C. Differences between means were evaluated by a t-test at the 5% level. Experiment 2 The plants sampled for this experiment had un- dergone > 90 treatments with the ABS solution. At the time of measurement the current needles, produced in spring 1989, had undergone at least 10 treatments with the surfactant. The main objective of this experiment was to assess the variation of whole plant transpiration and stomatal conductance in ABS-treated and control plant subjected to a condition of progres- sive and severe water stress. In June 1989, 8 control and 8 treated plants, both well-watered, were sampled for uniformity and subjected to drought treatment (water supply was interrupted between June 29 and July 21). The measure- ments were carried out as described in experi- ment 1, using 4 plants per group for destructive measurements (xylem water potential) and 4 other plants for non-destructive measurements (transpiration and stomatal conductance). The water potential was measured at dawn and the transpiration rate was assessed on a daily ba- sis. Stomatal conductance was measured at ir- regular intervals, when photosynthetic active ra- diation was > 1000 μmolm -2 s -1 . RESULTS Experiment 1 In the course of this experiment the early morning water potential (measured at 7:00 h am) ranged from a maximum be- tween -0.5 and -0.7 MPa on April 14 to a minimum between -1.8 and -2.6 MPa on July 14, and the plants experienced day- time water potentials as low as -3.6 MPa. Daily patterns of water potential, transpira- tion rates and stomatal conductances did not differ between the ABS-treated and the control plants. The physiological effect of the applied treatments may be better evaluated by considering the relationship between the measured variables. For instance, the variation of the whole plant transpiration as a function of the changing plant water status can be assessed by plotting the transpiration rates against the xylem water potential. This is shown in figure 2. The response of transpiration to water potential follows a characteristic pattern (fig 2). Transpiration rate was not affected by the water potential when this was be- tween -0.4 and -1.6 MPa. Indeed, within this range of water potential the transpira- tion rate varied from 10-90 mg m -2 s -1 , with most variation probably being deter- mined by the changing micrometeorologi- cal conditions within the glasshouse, where the relative humidity varied from 48-95% the air temperature from 11-32 °C and the solar radiation from 100-750 W m -2 during the measurement days. The water potential threshold which caused transpiration to decline may be set between -1.4 and -1.8 MPa. Indeed, be- low -1.8 MPa no transpiration rates > 10 mg m -2 s -1 were measured. It is worth not- ing that no different behaviours were dis- played by ABS-treated and control plants (fig 2). The stomatal conductance of current needles showed a wide range of variation, from zero to 0.9 cm s -1 . In order to relate the stomatal function to plant water status, conductance was plotted against the xylem water potential (fig 3). From this figure, it is evident that the stomatal conductance was not affected by the water potential when this was > -1.4 MPa, and decreased to zero when the water potential fell between -1.4 and -1.8 MPa. Even in this case, the ABS-treated and control plants behaved in the same way. The experiment described above was repeated using spruce seedlings from an Apennine population (Campolino, Tusca- ny), with basically the same results (data not shown). The net photosynthesis was measured on August 3, at 3 times of day. In plants displaying a water potential > -1.6 MPa a mean value of 2.0 μmol m -2 s -1 was as- sessed. The control and the ABS-treated plants displayed net photosynthesis values in the range 2.03-2.71 and 1.19-2.17 μmol m -2 s -1 , respectively; the observed differences were not statistically signifi- cant. The mean value of net photosynthe- sis fell to 0.35 μmol m -2 s -1 in plants exhib- iting a water potential < -1.6 MPa. The specific leaf area of current and old- er needles was measured on samples tak- en on August 3 1988 from control and ABS-treated plants. Mean values for cur- rent needles were in the range 39.1-40.4 cm 2 g -1 , and for older needles in the range 40.7-41.5 cm 2 g -1 . No statistically signifi- cant differences were found between ABS- treated and control plants. Experiment 2 During this experiment the plants experi- enced a pronounced condition of water stress. Indeed, the mean value of dawn water potential dropped from -0.2 MPa on 29 June to -3.8 MPa on 21 July. The tem- poral variation of dawn water potential, whole plant transpiration and stomatal con- ductance is given in figure 4 for the ABS- treated and the control plants. During the first week of drought dawn water potential was > -1.0 MPa; afterwards, water poten- tial decreased regularly. Similarly, the daily transpiration remained fairly constant, with values > 30 mg m -2 s -1 during the first week, suddenly declining when the dawn water potential dropped < -1.0 MPa and falling to zero when the water potential de- creased < -1.5 MPa. Stomatal conduc- tance displayed a similar pattern, and fell to zero when the dawn water potential was < -1.5 MPa. Even during this experiment the behav- iour of the ABS-treated and the control plants was the same. Indeed, no statistical- ly significant differences were found be- tween the physiological values displayed by the 2 groups of plants at the various dates. DISCUSSION AND CONCLUSIONS The experiments carried out made it possi- ble to study the water relations of spruce seedlings (both control and ABS-treated plants) exposed to periods of drought. In experiment 1, transpiration rates and stom- atal conductances were greatly reduced when the xylem water potential fell < -1.4 MPa. A threshold effect of the dawn water potential on daily transpiration rates and stomatal conductances was also found in experiment 2. These results confirm that in spruce species water stress becomes a re- ducing factor for transpiration and stomatal conductance when the water potential reaches some threshold value (Jarvis, 1976; Beadle et al, 1978; Havranek and Benecke, 1978). As far the main point addressed in the present work is concerned, ie the assess- ment of a possible effect of treatments on plant water relations, it is worth noting that the variation of transpiration rate and stom- atal conductance, as a function of the plant water status, was the same in ABS-treated and control plants. In particular, no lower transpiration rates and stomatal conduc- tances were observed in treated plants. Moreover, in treated plants the stomata closed regularly in response to the de- creasing xylem water potential. In other words, in the present experiment the ap- plied treatments did not alter stomatal functionality and decrease water vapour diffusion. No unregulated water loss from the needles was also assessed, since very low transpiration rates were measured af- ter stomatal closure. Furthermore, no evi- dence arose of a decreasing photosynthet- ic capacity in treated plants. That prolonged treatments with ABS did not alter plant water relations may be con- sidered as a rather surprising result. In- deed, only a few treatments with ABS, at the same concentration as those used in the present work, caused appreciable ef- fects on wax morphology, in particular ag- gregations of the fibrillar waxes and fis- sures in the epistomatal chamber in 5-yr- old Norway spruce seedlings (Raddi, 1990; personal communication). Furthermore, af- ter spraying young plants of Pinus pinea with a 40 g m -3 ABS solution, Gellini et al (1983) observed that about half the nee- dles were damaged after a single treat- ment with the percentage rising to 80% af- ter the application was repeated. After 35- 40 treatments with ABS solution at 50 g m -3 , a significant decrease in chlorophyll con- tent in 1-yr-old needles of Norway spruce seedlings was also observed (Heimler et al, 1989). . and -2.6 MPa on July 14, and the plants experienced day- time water potentials as low as -3.6 MPa. Daily patterns of water potential, transpira- tion rates and stomatal. specific leaf area. Picea abies / Pinaceae / Norway spruce / surfactant / pollution / transpiration / stomatal con- ductance / water potential Résumé — Relations hydriques. assess- ment of a possible effect of treatments on plant water relations, it is worth noting that the variation of transpiration rate and stom- atal conductance, as a